Heart disease is the single largest killer of Americans. Those that survive a heart attack have a higher risk of another attack heart failure, and stroke. Consequently, there is a need to develop approaches for repairing the heart and/improving the quality of life after a heart attack. The concept of regenerative medicine through the use of stem cells is gaining attention as a potential therapy for restoring cardiac function. Hurdles currently faced in the development of cellular therapies for the clinic are (1) the limited availability of cell surface markers for selecting pure cell populations and for tracking differentiation and (2) the lack of knowledge of how in vitro differentiation and in vivo development of cardiomyocytes correlate. The long term goal of this project is to correlate the function of a pure population of stem cells with a panel of defined cell surface markers. The major objective of the first granting period (K99) is to address the need for informative cell surface markers that are useful for characterizing specific cell types, stages, and lineages. I will apply the experience and knowledge gained from the research in the first granting period to understanding stem cell development in the second phase (ROO). The aims of the ROO phase are designed to develop an understanding of whether the molecular events during in vitro differentiation mimic those during in vivo development of cardiomyocytes. My long term career goal is to lead an independent research group that utilizes analytical technologies to bridge basic science research and clinical applications of stem cells. This research plan is specifically designed to take advantage of my analytical expertise while providing a relevant biological context to acquire new knowledge and skills necessary maximize the use of analytical methods within the context of biology. The training environment at Johns Hopkins is especially suited to this research due to the access to state-of-the art instrumentation, mentors with specialized expertise, and the forum to support discussion of clinically relevant research. Relevance: The focus of the current proposal is to identify specific cell surface protein markers that will aid clinicians and researchers in understanding the mechanisms of stem cell differentiation, both within the animal and in culture. Accessible markers for the specific selection, enrichment, and tracking of stem cell populations appropriate for cardiovascular regenerative medicine will be generated. (End of Abstract)